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Improving the intestinal microbiota using probiotics, prebiotics, and synbiotics has attracted attention as a method of disease prevention and treatment. This is the first study to discuss the effects of food intake on the intestinal microbiota using a large Japanese intestinal microbiota database. Here, as a case study, we determined changes in the intestinal microbiota caused by ingestion of a processed natto food containing B. subtilisvar. natto SONOMONO spores, SONOMONO NATTO POWDER CAPSULESTM, by analyzing 16S rRNA sequence data generated using next-generation sequencing techniques. The results showed that the relative abundance of Bifidobacterium and Blautia as well as the relative abundance of Bifidobacterium were increased in males and females in the ingesting group, respectively. Additionally, the effects of SONOMONO NATTO POWDER CAPSULESTM intake on Bifidobacterium and Blautia abundance depended on the relative abundance of Bifidobacterium at baseline. Finally, analysis of a large Japanese intestinal microbiota database suggested that the bacterial genera that fluctuated with the ingestion of SONOMONO NATTO POWDER CAPSULESTM may be associated with lifestyle-related diseases such as diabetes.

This study aimed to improve the nutritional value of soybean meal (SBM) by solid-state fermentation (SSF) using Bacillus subtilis natto (B. s. natto) to overcome the limitations of SBM usage in aquafeed. The response surface methodology (RSM) was employed to explore the relationships of fermentation conditions, such as temperature, time, water-substrate ratio, and layer thickness, on the degree of protein hydrolysis (DH) and the crude protein (CP) content. The optimum conditions for achieving the higher DH (15.96%) and CP (55.76%) were 43.82 °C, 62.32 h, 1.08 of water-substrate ratio, and a layer thickness of 2.02 cm. CP and DH in the fermented soybean meal (FSM) increased by 9.8% and 177.1%, respectively, and crude fiber decreased by 14.1% compared to SBM. The protein dispersibility index (PDI) decreased by 29.8%, while KOH protein solubility (KPS) was significantly increased by 17.4%. Flavonoids and total phenolic acid content in FSM were increased by 231.0% and 309.4%, respectively. Neutral protease activity (NPA) also reached a high level (1723.6 U g−1). Total essential amino acids (EAA) in FSM increased by 12.2%, higher than the 10.8% increase of total non-essential amino acids (NEAA), while the total free amino acids content was 12.76 times higher than that of SBM. Major anti-nutritional factors in SBM were significantly reduced during the process, and almost all SBM protein macromolecules were decomposed. Together with the cost-effectiveness of SSF, B. s. natto-fermented SBM products have great potential to improve the plant composition and replace high-cost ingredients in aquafeed, contributing to food security and environmental sustainability.

Although diverse immunomodulatory reactions of probiotic bacteria have been reported, this effect via Bacillus subtilis natto remains unclear, despite its long consumption history in Japan and usage in Natto production. Hence, we performed a comparative analysis of the immunomodulatory activities of 23 types of B. subtilis natto isolated from Natto products to elucidate the key active components. Among the isolated 23 strains, the supernatant from B. subtilis strain 1 fermented medium showed the highest induction of anti-inflammatory IL-10 and pro-inflammatory IL-12 in THP-1 dendritic cells (THP-1 DC) after co-incubation. We isolated the active component from strain 1 cultured medium and employed DEAE-Sepharose chromatography with 0.5 M NaCl elution for fractionation. IL-10-inducing activity was specific to an approximately 60 kDa protein, GroEL, which was identified as a chaperone protein and was significantly reduced with anti-GroEL antibody. Differential expression analysis of strains 1 and 15, which had the lowest cytokine-producing activity, showed a higher expression of various genes involved in chaperones and sporulation in strain 1. Furthermore, GroEL production was induced in spore-forming medium. The present study is the first to show that the chaperone protein GroEL, secreted by B. subtilis natto during sporulation, plays a crucial role in IL-10 and IL-12 production in THP-1 DC.

Lentinan has important applications in the food and medicine fields. Fermenting Lentinula edodes with Bacillus subtilis natto increased the lentinan extraction yield by 87.13% and greatly altered the molecular structure and antioxidant activity of lentinan. The uronic acid content in the lentinan molecular structure increased from 2.08% to 4.33%. The fermentation process did not affect the monosaccharide composition of lentinan, comprised of more than 90% glucose residues. Fermentation significantly reduced the molecular weight of lentinan and altered its apparent structure. The water solubility of fermented lentinan was increased by 165.07%, and the antioxidant activity was significantly improved. Fermentation using soybean as a substrate may be beneficial for enhancing the activity of Bacillus subtilis natto and producing lentinan with different molecular weights.

Bacillus subtilis (natto) is generally regarded as a safe bacterium and used as a host for the production of several materials. However, genetic engineering of B. subtilis (natto) is not well established because of poor DNA delivery methods and the lack of a standard strain for the aim. Here, we developed a genetic delivery tool in B. subtilis (natto) using the pLS20 conjugational plasmid (65 kbp). Transmission of pLS20 from B. subtilis 168 to wild-type B. subtilis (natto) did not occur via established mating protocols. We isolated B. subtilis (natto) mutants showing dramatically increased recipient activity. Whole-genome sequence analyses revealed three common alterations: mutations in the restriction endonuclease gene and in the methyl-accepting chemotaxis protein gene, and a 43-kbp deletion at the genome replication termination locus. A representative strain named NEST116 was generated as the first B. subtilis (natto) strain suitable for exploring pLS20-based genetic engineering.

Fermented foods are defined as foods or beverages produced through controlled microbial growth, and the conversion of food components through enzymatic action. In recent years, fermented foods have undergone a surge in popularity, mainly due to their proposed health benefits. The aim of this review is to define and characterise common fermented foods (kefir, kombucha, sauerkraut, tempeh, natto, miso, kimchi, sourdough bread), their mechanisms of action (including impact on the microbiota), and the evidence for effects on gastrointestinal health and disease in humans. Putative mechanisms for the impact of fermented foods on health include the potential probiotic effect of their constituent microorganisms, the fermentation-derived production of bioactive peptides, biogenic amines, and conversion of phenolic compounds to biologically active compounds, as well as the reduction of anti-nutrients. Fermented foods that have been tested in at least one randomised controlled trial (RCT) for their gastrointestinal effects were kefir, sauerkraut, natto, and sourdough bread. Despite extensive in vitro studies, there are no RCTs investigating the impact of kombucha, miso, kimchi or tempeh in gastrointestinal health. The most widely investigated fermented food is kefir, with evidence from at least one RCT suggesting beneficial effects in both lactose malabsorption and Helicobacter pylori eradication. In summary, there is very limited clinical evidence for the effectiveness of most fermented foods in gastrointestinal health and disease. Given the convincing in vitro findings, clinical high-quality trials investigating the health benefits of fermented foods are warranted.

Atherosclerosis is a chronic inflammatory disease characterized by lipid accumulation and immune dysregulation, including expansion of pro-inflammatory monocytes and effector T cells, alongside reduced regulatory T cells (Tregs). Bacillus subtilis natto, a spore-forming probiotic, has shown anti-atherosclerotic effects, though its systemic immunomodulatory mechanisms remain unclear. In this study, we employed an AAV-mPCSK9-induced murine atherosclerotic model to investigate the effects of daily B. subtilis natto NTU-18 administration over 16 weeks. High-dimensional flow cytometry using two 13-marker panels enabled longitudinal profiling of 18 peripheral immune cell subsets across lymphoid and myeloid compartments. While no significant changes in serum cholesterol and mild decrease of body weight were observed, B. subtilis natto NTU-18-treated mice presented a significant reduction in aortic lesion area compared to PBS-treated controls. Immune profiling revealed a transient expansion of peripheral myeloid cells and CD44⁺ trained CD8⁺ T cells, followed by increased frequencies of naïve CD8⁺ T cells and reduced central/effector memory subsets at longer time point treatment. In the CD4⁺ T cell compartment, a transient increase in trained cells was accompanied by a sustained enrichment of CD25⁺CD4⁺ Tregs throughout the daily B. subtilis natto NTU-18 treatment. In contrast, no significant differences were observed in Ly6C⁻ or Ly6C⁺ monocytes, neutrophils, or eosinophils. These findings suggest that B. subtilis natto NTU-18 attenuates atherosclerosis progression not through lipid lowering or broad myeloid modulation, but via targeted reprogramming of peripheral T cell responses. This work provides mechanistic insight into the immunotherapeutic potential of B. subtilis natto NTU-18 in atherosclerosis prevention and treatment. Key points • B. subtilis natto NTU-18 significantly reduces aortic plaque burden in atherosclerotic mice without affecting serum cholesterol levels . • B. subtilis natto NTU-18 induces transient immune remodeling, marked by early expansion of trained CD8⁺ and CD4⁺ T cells, followed by increased naïve and regulatory T cells . • The atheroprotective effect is primarily mediated through adaptive immunity as myeloid subsets remain unchanged throughout B. subtilis natto NTU-18 treatment .

Fructooligosaccharides and levan have a wide range of applications in the food industry due to their physiological and functional properties. The enzymatic synthesis of these molecules exhibits great advantages when compared with microbial fermentation. In this study, the production of levansucrase from Bacillus subtilis natto and its utilization in fructooligosaccharides and levan syntheses using different reaction conditions were described. The best condition for levansucrase production was 420.7 g L−1 of sucrose at pH 7.0, which reached 23.9 U ml−1 of transfructosylation activity. In a bioreactor, the highest production of fructooligosaccharides was 41.3 g L−1 using a medium containing 350 g L−1 sucrose at 35 °C for 36 h. The enzymatic synthesis of levan resulted in 86.9 g L−1 when conditions similar to those used for fructooligosaccharides synthesis were applied. These results indicate that the levansucrase from B. subtilis natto could be applied for the co-production of fructooligosaccharides and levan, which are biomolecules that have health benefits and are used successfully in the food industry.

Bacillus subtilis natto is a GRAS bacterium. Nattokinase, with fibrinolytic and antithrombotic activities, is one of the major products of this organism. It is being gradually recognized that B. subtilis natto can also be used as a biosynthetic strain for vitamin K2, which has phenomenal benefits, such as effects in the prevention of cardiovascular diseases and osteoporosis along with antitumor effects. Knocking out of the aprN gene by homologous recombination could improve the redox potential and slightly increase the concentration of MK-7. By detecting the change in redox potential during the growth of B. subtilis natto, a good oxygen supply and state of the cell membrane were found to be beneficial to vitamin K2 synthesis. A two-step RSM was used to optimize the operation parameters and substrate concentration in the new residue-free fermentation culture. The optimal conditions for the residue-free medium and control were determined. The optimum concentrations of soybean flour, corn flour, and peptone were 78.9, 72.4, and 24.8 g/L, respectively. The optimum rotational speed and volume of the culture medium using a shaking flask were 117 rpm and 10%, respectively. The state and composition of the cell membranes were more stable when engineered bacteria were cultured in this residue-free fermentation medium. Finally, the concentration of MK-7 increased by 37% to 18.9 mg/L, and the fermentation time was shortened by 24 h.

Background Nattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. In order to increase nattokinase yields, fermentation conditions, strains, cultivation media, and feeding strategies have been optimized. Nattokinase has been expressed using several heterologous expression systems. Pichia pastoris heterologous expression system was the alternative. Results This report aimed to express high levels of nattokinase from B. subtilis natto (NK-Bs) using a Pichia pastoris heterologous expression system and assess its fibrinolytic activity in vivo. Multicopy expression strains bearing 1–7 copies of the apr N gene were constructed. The expression level of the target protein reached a maximum at five copies of the target gene. However, multicopy expression strains were not stable in shake-flask or high-density fermentation, causing significant differences in the yield of the target protein among batches. Therefore, P. pastoris bearing a single copy of apr N was used in shake-flask and high-density fermentation. Target protein yield was 320 mg/L in shake-flask fermentation and approximately 9.5 g/L in high-density fermentation. The recombinant nattokinase showed high thermo- and pH-stability. The present study also demonstrated that recombinant NK-Bs had obvious thrombolytic activity. Conclusions This study suggests that the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of nattokinase.